T.L. Zimmer

The presence and possible role of phosphopantothenic acid in gramicidin S synthetase

Orn + Leu [5]. In agreement with this finding it was shown independently by Gevers, Kleinkauf and Lipmann [4] and FrQyshov, Zimmer and Laland [3] that di-, tri-, tetra-, and pentapeptides all having D-phenylalanine at the N-terminal end aswell as all five amino acids in gramicidin S are bound to the enzyme through thio- ester linkages. The possible presence of 4’-phosphopantetheine in gramicidin S synthetase occurred to us some time ago when evidence for the presence of acid stable and alkali labile enzyme intermediates was obtained [ 1, 21. Preliminary analysis of a gramicidin S synthetase preparation, designated fraction 5 and *

The nature of the enzyme bound intermediates in gramicidin s biosynthesis

Previous work [ 1,2] indicated that during synthesis, intermediates are covalently bound to grarnicidin S synthetase. Furthermore, the synthesis starts with phenylalanine and the sequence of addition of amino acids is Phe-Pro-Val-Orn [2]. When the present work was completed, a paper by Gevers, Kleinkauf and Lipmann [3] appeared which indicated that all fne amino acids and the intermediate peptides D-Phe-L-Pro, D-Phe-L-Pro-L-Val, D-Phe-LPro-L-Val-L-Om, and D-Phe-L-Pro-L-Val-LOrn-L-Leu are bound covalently to the synthetase, most probably through thioester linkages. This work presents additional evidence for this view. The present results also show that there is a marked difference in the stability of the linkages of ornithine and D-PheL-Pro to the protein towards ethanolHC1 compared to that of the other covalently bound intermediates. Using a different method to that of Gevers et al. [3] no evidence for peptides longer than the pentapeptide was found. This finding supports the view that gramicidin S is formed by head to tail condensation of activated pentapeptides

[43] Gramicidin S synthetase

Publisher Summary This chapter discusses the assay and purification procedure of gramicidin S synthetases. The following enzymic activities may be used for assaying gramicidin S synthetase: (1) synthesis of gramicidin S; (2) amino acid-dependent ATP- 32 PPi exchange; (3) amino acid-dependent ATP-[ 14 C] AMP exchange; (4) thioesterbonding of the individual amino acids, (5) ATP-dependent racemization of phenylalanine. Purification procedure involves cultivation of the microorganism, preparation of crude extract, streptomycin sulfate precipitation, ammonium sulfate precipitation, and chromatography on DEAE sephadex A-50, chromatography on sephadex G-200 alternative method or the separation of light and heavy enzyme: affinity chromatography. The biosynthesis of gramicidin S is the result of the concomitant functioning of a large number of catalytic activities. For instance, in the case of the heavy enzyme it has been estimated that about 18–20 different catalytic activities are involved. During the purification of gramicidin S synthetases that gives an almost homogeneous preparation of heavy enzyme, some 97% of its ability to synthesize the antibiotic is lost. The loss is particularly great during fractionation on the DEAE Sephadex G-50 and Sephadex G-200. Of the many catalytic activities involved in the biosynthesis, the catalytic activities responsible for amino acid activation are remarkably stable.

A rapid method for the preparation of pure heavy enzyme of gramicidin S synthetase.

Gramicidin S synthetase consists of the light enzyme (mol. wt 100 000) and the heavy enzyme (mol. wt 280 000) which both have been isolated in essentially pure form [l-3] . The present report describes a rapid, simple and reproducible procedure for the isolation of pure heavy enzyme in high yield. Evidence is also presented that the heavy enzyme is not split into subunits by standard methods suggesting that the enzyme contains only one polypeptide chain or that several polypeptide chains may be covalently linked by bonds other than disulphide bridges.

Replacement of phenylalanine in gramicidin S by other amino acids

This decapeptide is synthesized by gramicidin S synthetase which consists of two enzymes, the light and the heavy [I]. The light enzyme which is also a racemase, activates and thioesterbinds phenylalanine. The growth of the peptide chain is initiated by transfer of the thioester-bound D-phenylalanyl group to the heavy enzyme. This enzyme activates and thioesterbinds L-proline, Gvaline, L-ornithine and L-leucine and catalyzes the formation of the peptide bonds in gramicidin S. In the past, substitution of phenylalanine in gramicidin S by other amino acids using gramicidin S synthetase has been claimed to take place. For instance it has been reported that p-fluorophenylalanine and fl-thienylalanine will substitute [2]. However, in neither case have the cyclic decapeptides been identified chromatographically and separated from gramicidin S. Furthermore, there is conflicting evidence on whether or not tyrosine can replace phenylalanine [2,3]. It has also been claimed that tryptophan could not replace this amino acid [3]. We have reexamined these reports and present evidence that p-fluoro-, p-chloro-, p-bromophenylalanine, /3-thienylalanine, tyrosine and tryptophan can replace phenylalanine. Evidence for the formation of a hybrid decapeptide containing one residue of tyrosine and one of phenylalanine is also presented. In addition, cyclohexylalanine, phenylglycine and a-methyl phenylalanine have been examined for their ability to substitute for phenylalanine. None of these could substitute in the synthesis. However, it was found that in the presence of cyclohexylalanine and proline only, synthesis is initiated since the dipeptide cyclohexylalanylproline is produced. If valine and leucine were present, the formation of the dipeptide was inhibited. This probably explains why no synthesis of the cyclic decapeptide takes place in a complete incubation mixture.

An unexpected activation and thioester binding of D- and L-phenylalanine by the heavy enzyme of gramicidin S synthetase

Gramicidin S synthetase consists of the light (100 000 dalton) and the heavy (280 000 dalton) enzyme. The light enzyme activates Phe and initiates synthesis by transferring the thioester-bound D-phenylalanyl group from the light to the heavy enzyme which activates L-Pro, LVal, L-Orn and L-Leu and catalyzes the remaining (about 19) of the reactions required for synthesis of the cyclic decapeptide [ 11. Recently, using heavy enzyme [ 21 not contaminated by the light enzyme, we have unexpectedly found that it activates and thioesterbinds Land D-Phe. In the past the ATP-PP, exchange reaction in the presence of Phe has been used as a test for the presence of light enzyme in the heavy. The present results show that this is not necessarily correct. The thiol site involved in the binding of Land D-Phe to the heavy enzyme in the absence of light enzyme does not participate in gramicidin S synthesis since the D-[ 14C]Phe thioesterbound to the heavy enzyme after incubation with labelled phenylalanine could not be incorporated into gramicidin S. It is therefore not the site which has been suggested to be involved when the D-phenylalanyl group is transferred from the light to the heavy enzyme [3].

Non-ribosomal biosynthesis of linear gramicidins.

It is now well established that the biosynthesis of the cyclic peptides gramicidin S and tyrocidine [2] are synthesized independently of ribosomes and it has been suggested that the linear gramicidins are synthesized by a similar mechanism [3] . However, since no cell free system for the study of the synthesis of this peptide is yet available, it was considered of interest to investigate in whole cells if the synthesis was independent of ribosomes. In the present work the synthesis of the linear gramicidins was studied in whole cells in the presence of chloramphenicol. The results clearly indicate that the synthesis is independent of ribosomes.